Cells interact with structurally distinct types of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What are the differences in cell adhesive structures and biological responses to two-dimensional (2D) versus three-dimensional (3D) matrices, as well as between different types of 3D matrices characteristic of different in vivo microenvironments? 2. What signal transduction mechanisms control cell behavior in different 3D microenvironments? We previously published on the importance of a 3D environment for a variety of cell biological processes including fibroblast migration. This latter paper reported the existence of distinctive cell-matrix adhesions in 3D. However, 3D environments can differ substantially: cells in different locations or stages of development in vivo interact with biochemically and structurally distinct matrices, e.g. a matrix rich in fibronectin fibrils during early craniofacial neural crest migration versus collagen-rich matrices in adult connective tissues. Consequently, we hypothesized that biochemical composition can alter cell migration, the actin cytoskeleton, and/or types of cell adhesion structures. We have quantified these parameters to establish direct comparisons for normal, non-tumor cells migrating in four different, widely used 3D matrices, i.e., collagen I and fibrin gels, cell-derived 3D matrix, and basement membrane extract (Matrigel). We found that cells in the different matrices display distinctive morphologies of cell adhesions, differing cell spread area, and significantly different capacities for rapid cell migration on/in 2D versus 3D matrices. Our findings establish that one cannot merely study cell adhesion and migration generically in 3D, but instead that the choice of the particular matrix environment plays an important role the cellular phenotype. This work underscores the importance of choosing the appropriate materials for biomimetic scaffolds and microenvironments to be used for tissue engineering. A recently published study raised serious questions about whether well-defined cell-matrix adhesions actually exist in 3D. Resolving this basic question has important implications for the relevance of the thousands of papers previously published characterizing cell adhesions in cell culture. We found that we could, in fact, readily identify and quantify cell-matrix adhesions in all four 3D matrices that we tested: collagen I, fibrin, cell-derived matrix, and basement membrane extract. The total cell surface area devoted to integrin-containing cell adhesions was similar in 2D and 3D conditions. In separate studies, we could also readily detect dynamic 3D matrix adhesions in living human fibroblasts migrating in either collagen gels or cell-derived matrix using GFP (green fluorescent protein)-tagged paxillin. Our findings are complementary to those of the Horwitz laboratory in refuting claims about the absence of discrete cell-matrix adhesions in 3D. Nevertheless, we also observed interesting discrepancies in the composition of adhesions in very soft collagen gels which suggest that vinculin, a mechanosensitive linker molecule, can be decreased in adhesions depending on the physical properties of the matrix, even though integrin-based adhesions remain. We explored this concept in depth by comparing primary images in the entire published literature on 3D cell-matrix interactions and adhesions, as detailed in a review currently in press. We propose a conceptual model in which integrin-based cell-matrix adhesions are widespread in 3D environments, but their cell adhesion components can vary depending on local matrix physical properties and cellular mechanosensing. We are currently testing whether classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environment found in tissues. We are using tissue explants and in vitro models of 3D extracellular matrix consisting of cell-derived matrix and collagen gels to mimic different types of complex tissue structure, and high-resolution live-cell imaging to visualize intracellular signaling. Our goal is to determine the intracellular organization of signaling pathways in migrating cells in order to understand the mechanisms and regulation of cell motility in structurally complex 3D environments. We are focusing particularly on searching for unique modes of migration and signaling through Rho GTPases within different extracellular matrix environments.